The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The use of long life-time emitting lanthanide(III) chelate labels or probes together with time-resolved fluorometry in detection provides a method to generate sensitive bioaffinity assays. Indeed, time-resolved fluorescence based on lanthanide(III) chelates has become a successful detection technology, and it has been used in in vitro diagnostics for over two decades. Time-resolved fluorescence quenching assays based on energy transfer from a lanthanide(III) chelate to a nonfluorescent quencher have been applied in various assays of hydrolyzing enzymes as well as for nucleic acid detection. The different photochemical properties of europium, terbium, dysprosium and samarium chelates even enable the development of multiparametric homogenous assays.
Stable luminescent lanthanide(III) chelates consist of a ligand with a reactive group for covalent conjugation to bioactive molecules, an aromatic structure, which absorbs the excitation energy and transfers it to the lanthanide ion and additional chelating groups such as carboxylic or phosphonic acid moieties and amines. Unlike organic chromophores, these molecules do not suffer from Raman scattering or concentration quenching. This allows multilabeling and development of chelates bearing several light absorbing moieties.
A luminescent lanthanide(III) chelate has to fulfill several requirements a) the molecule has to be photochemically stable both in the ground and excited states, b) the molecule has to be kinetically stable, c) the molecule has to be chemically stable, d) the excitation wavelength has to be as high as possible, preferably over 330 nm, e) the molecule must have a high excitation coefficient in the excitation wavelength, f) the energy transfer from the ligand to the central ion has to be efficient, g) the luminescence decay time has to be long, h) the chelate should be readily soluble in water, i) the bioactive molecules have to retain their affinities after the coupling to the lanthanide chelate.
A number of attempts have been made to tune the photophysical properties of the chelate labels suitable for time-resolved fluorometric applications. These include e.g. stable chelates composed of derivatives of pyridines [U.S. Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, U.S. Pat. No. 4,459,186, EP 0770610], bipyridines [U.S. Pat. No. 5,216,134], terpyridines [U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,324,825] or various phenolic compounds [U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191] as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563], macrocyclic cryptates [U.S. Pat. No. 4,927,923, EP 493745A] and macrocyclic Schiff bases [EP 369000] have been disclosed.
It has been shown that an europium(III) chelate based on 1,4,7-triazacyclononane tethered to three phenylethynylpyridinyl chromophores has good luminescence properties: its luminescence yield (εΦ) is significantly higher than that of the chelate constructed from a single chromophore [Helv. Chim. Acta, 1996, 79, 789]; also its kinetic and thermodynamic stabilities are high. However, the chelate disclosed is not suitable for biomolecule derivatization because it lacks the reactive group required for conjugation. Furthermore, the ethynyl groups are susceptible to photobleaching, which is a problem especially in applications based on fluorescence microscopy. The alkynyl groups may also react with additives needed in in vitro assays, especially the highly nucleophilic azide ion. Later, the above mentioned problems have been solved by substituting the phenylethynyl groups with furyl and trismethoxyphenyl subunits giving rise to luminescent chelates with europium and samarium as well as with terbium and dysprosium, respectively [U.S. patent application Ser. No. 11/004,061; U.S. patent application Ser. No. 10/928,143].
The azamacrocycles disclosed still have some drawbacks: the aromatic chromophores decrease their solubility to water. Also, the excitation maxima of the furyl derivatives are only somewhat over 300 nm; a higher excitation wavelength would be desirable while developing simpler and less expensive detection instruments and reduce the significance of the background luminescence signal. Furthermore, shorter wavelengths are absorbed by biological materials such as nucleic acids and aromatic amino acids.
Azamacrocycles tethered to long wavelength sensitizers, such as aromatic heterocycles, have also been proposed [e.g. U.S. Pat. No. 6,344,360, WO2006/039505, WO2007/055700, J. Chem. Soc. Perkin Trans 2, 2002, 348]. However, the emission spectrum of chelates of this type is often divided into several peaks. This, in turn causes problems: a) the quantum yield is relatively low when narrow filters have to be used, such as in multilabel assays, b) the additional emission bands cause background signal in applications based on time-resolved fluorescence energy transfer, c) the intensive long wavelength emission lines limit the use of NIR acceptors.
Although organic chelators and their substituents have a significant effect on the photophysical properties of lanthanide(III) chelates, no general rules for the estimation of these effects are available. It has been proposed in U.S. Pat. No. 4,761,481 that electron releasing substituents in the aromatic moiety of phenyl and naphthyl substituted 2,6-[N,N-di(carboxyalkyl)aminoalkyl]pyridines have advantageous effects on the photophysical properties on their chelates with lanthanide ions. However, no experimental evidence was given. Later it has been shown that this is the case with various terbium(III) and dysprosium(III) chelates [U.S. patent application Ser. No. 11/004,061] but the corresponding europium(III) chelates are practically non-luminescent [Hemmilä et al., J. Biochem. Biophys. Methods, 1993, 26, 283]. Furthermore, in contrast to that proposed in U.S. Pat. No. 4,761,481 it has been shown that lanthanide chelates with electron releasing amino substituent in the aromatic moieties have low quantum yields [Takalo et al., Helv. Chim. Acta, 1993, 76, 877].
In several applications, covalent conjugation of the chelate to bioactive molecules is required. Most commonly, this is performed in solution by allowing an amino or mercapto group of a bioactive molecule to react with isothiocyanato, maleimido or N-hydroxysuccinimido derivatives of the label [Fichna, J., Janecka, A., Bioconjugate Chem., 2003, 14, 3]. Since in almost all biomolecule labelings the reaction is performed with an excess of an activated label, laborious purification procedures cannot be avoided. Especially, when the attachment of several label molecules, or site-specific labeling in the presence of several functional groups of similar reactivity is required, the isolation and characterization of the desired biomolecule conjugate is extremely difficult, and often practically impossible.
The biomolecule conjugates used in many applications, such as homogenous quenching assays, have to be extremely pure, since even small amounts of fluorescent impurities considerably increase the luminescence background and reduce the detection sensitivity. Thus, it is highly desirable to perform the conjugation of biomolecules on solid phase, since most of the impurities can be removed by washings while the biomolecule is still anchored to the solid support, and once released into the solution, only one chromatographic purification is required.
Solution phase labeling of large biomolecules, such as proteins, cannot be avoided. In these cases, the labeling reaction has to be as selective, and the purification of the biomolecule conjugates as effective as possible.